<Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Elongin BC complex is essential for cell proliferation and chromosome condensation and segregation during mitosis and meiotic division II

The ubiquitin-mediated protein degradation system is involved in a wide variety of cellular functions. The RING-H2 finger protein RBX1 is a common subunit of Cullin-based ubiquitin ligases. Caenorhabditis elegans RBX1 and CUL2 are essential for regulating chromosome condensation and segregation during mitosis and meiosis and are also critical for cell proliferation. Here, we demonstrate that Elongin B (ELB1) and C (ELC1) form a stable complex, and that depletion of either gene product by RNA-mediated interference (RNAi) causes pronounced defects in the second meiotic division. Embryos and adults that escape meiotic arrest have several irregular phenotypes. These include defects in mitotic chromosomal condensation and segregation, pronuclear rotation, and germ cell proliferation, abnormal cortical protrusion, and accumulation of the cyclin-dependent kinase inhibitor CKI1. All these defects are consistent with those found after depletion of CUL2. In addition, direct interaction between ELC1 and CUL2 is revealed by bacterial two-hybrid analysis. Thus, the RBX1/CUL2/ELC1/ELB1 complex acts as an E3 ubiquitin ligase in C. elegans and is essential for diverse functions relevant to chromosomal dynamics and cell cycle control.     

WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs and is caused by mutations targeting either the TSC1 or TSC2 genes. TSC1 and TSC2 proteins form a functionally interdependent dimeric complex. Phosphorylation of either TSC subunit by different kinases regulates the function of TSC and represents a major mechanism to integrate various signals into a centralized cell growth pathway. The majority of disease-associated mutations targeting either TSC1 or TSC2 results in a substantial decrease in protein level, suggesting that protein turnover also plays a critical role in TSC regulation. Here we report that TSC2 protein binds to FBW5, a DDB1-binding WD40 (DWD) protein, and is recruited by FBW5 to the DDB1-CUL4-ROC1 E3 ubiquitin ligase. Overexpression of FBW5 or CUL4A promotes TSC2 protein degradation, and this is abrogated by the coexpression of TSC1. Conversely, depletion of FBW5, DDB1, or CUL4A/B stabilizes TSC2. Ddb1 or Cul4 mutations in Drosophila result in Gigas/TSC2 protein accumulation and cause growth defects that can be partially rescued by Gigas/Tsc2 reduction. These results indicate that FBW5-DDB1-CUL4-ROC1 is an E3 ubiquitin ligase regulating TSC2 protein stability and TSC complex turnover.     

Caenorhabditis elegans RBX1 is essential for meiosis, mitotic chromosomal condensation and segregation, and cytokinesis

BACKGROUND: The RING-H2 finger protein RBX1 (ROC1/HRT1) is a common subunit of SKP1-CDC53/CUL1-F-box (SCF), other cullins and von Hippel-Lindau (VHL) tumour suppressor E3 ubiquitin ligase complexes. RBX1 protein sequences are highly conserved in various species, including yeasts, Drosophila melanogaster, mice and humans. In Saccharomyces cerevisiae, RBX1 is essential for the G1/S transition. RESULTS: Caenorhabditis elegans RBX1 is strongly expressed in early embryos and in the gonad, including meiotic cells. Depletion of RBX1 by RNA-mediated interference (RNAi) caused pronounced defects in the first meiotic division. Several irregular phenotypes were identified in embryos that escaped from meiotic arrest: defects in mitotic chromosomal condensation and segregation, abnormal chromosome bridges, giant nuclei, abnormal cortical protrusion, multinucleate cells and defects in germ cell proliferation. Moreover, histone H3 phosphorylation at Ser10 and Ser28 was significantly reduced in these embryos. The histone H3 phosphorylation defect of embryos was rescued by the additional depletion of protein phosphatase 1 (GLC7alpha/beta) by RNAi. CONCLUSION: These results indicate that the RBX1 protein participates in diverse functions relevant to chromosome metabolism and cell cycle control.     

De novo variants in RHOBTB2, an atypical Rho GTPase gene,cause epileptic encephalopathy

By whole exome sequencing, we identified three de novo RHOBTB2 variants in three patients with epileptic encephalopathies (EEs). Interestingly, all three patients showed acute encephalopathy (febrile status epilepticus), with magnetic resonance imaging revealing hemisphere swelling or reduced diffusion in various brain regions. RHOBTB2 encodes Rho‐related BTB domain‐containing protein 2, an atypical Rho GTPase that is a substrate‐specific adaptor or itself is a substrate for the Cullin‐3 (CUL3)‐based ubiquitin ligase complex. Transient expression experiments in Neuro‐2a cells revealed that mutant RHOBTB2 was more abundant than wild‐type RHOBTB2. Coexpression of CUL3 with RHOBTB2 decreased the level of wild‐type RHOBTB2 but not the level of any of the three mutants, indicating impaired CUL3 complex‐dependent degradation of the three mutants. These data indicate that RHOBTB2 variants are a rare genetic cause of EEs, in which acute encephalopathy might be a characteristic feature, and that precise regulation of RHOBTB2 levels is essential for normal brain function.     

Bood POZ containing gene type 2 is a human counterpart of yeast Btb3p and promotes the degradation of terminal deoxynucleotidyltransferase

Bood POZ containing gene type 2 (BPOZ-2) is involved in the growth suppressive effect of the phosphatase and tensin homologue (PTEN). We showed that BPOZ-2 is a human counterpart of yeast Btb3p, which is a putative adaptor for Pcu3p-based ubiquitin ligase. BPOZ-2 bound to E3 ligase CUL3 in vitro and in vivo . BPOZ-2 itself was ubiquitinated through the CUL3-based E3 ligase mainly within the nucleus and degraded by the 26S proteasome. Although BPOZ-2 was mainly expressed within the cytoplasm, it accumulated within the nucleus in the presence of the specific 26S proteasome inhibitor MG132. BPOZ-2 may be recruited to the nucleus from the cytoplasm. Terminal deoxynucleotidyltransferase (TdT) was detected as a BPOZ-2-binding protein using a yeast two-hybrid system by screening a human thymus cDNA library. TdT, BPOZ-2, and CUL3 formed a ternary complex in vivo . TdT was ubiquitinated only within the nucleus and degraded by the 26S proteasome. The ubiqutination or degradation of TdT was markedly promoted by co-expression of BPOZ-2 and CUL3 or BPOZ-2 in 293T cells, respectively.     

Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution

The Keap1–Nrf2 regulatory pathway plays a central role in the protection of cells against oxidative and xenobiotic damage. Under unstressed conditions, Nrf2 is constantly ubiquitinated by the Cul3–Keap1 ubiquitin E3 ligase complex and rapidly degraded in proteasomes. Upon exposure to electrophilic and oxidative stresses, reactive cysteine residues of Keap1 become modified, leading to a decline in the E3 ligase activity, stabilization of Nrf2 and robust induction of a battery of cytoprotective genes. Biochemical and structural analyses have revealed that the intact Keap1 homodimer forms a cherry-bob structure in which one molecule of Nrf2 associates with two molecules of Keap1 by using two binding sites within the Neh2 domain of Nrf2. This two-site binding appears critical for Nrf2 ubiquitination. In many human cancers, missense mutations in KEAP1 and NRF2 genes have been identified. These mutations disrupt the Keap1–Nrf2 complex activity involved in ubiquitination and degradation of Nrf2 and result in constitutive activation of Nrf2. Elevated expression of Nrf2 target genes confers advantages in terms of stress resistance and cell proliferation in normal and cancer cells. Discovery and development of selective Nrf2 inhibitors should make a critical contribution to improved cancer therapy.     

Deletion of Herp facilitates degradation of cytosolic proteins

Although intracellular stresses are believed to be involved in the process of neurodegeneration, it is not fully understood how one stress/stress response affects another. Herp is an endoplasmic reticulum (ER)‐located membrane protein proposed to function in ER‐associated degradation (ERAD). Herp is strongly induced by ER stress but rapidly degraded by proteasome. To elucidate the effect of Herp expression on proteolytic stress caused by impairment of the ubiquitin‐proteasome system (UPS), we utilized 293T Herp knockdown (KD) cells and F9 Herp knockout cells. Knockdown of Herp gene unexpectedly facilitated the degradation of Parkinson’s disease‐associated cytosolic proteins such as α‐synuclein and its binding partner, synphilin‐1, and improved cell viability during proteasomal inhibition. A similar tendency was observed in F9 Herp knockout cells transfected with synphilin‐1. Herp temporarily bound to α‐synuclein, synphilin‐1 and the E3 ligase SIAH1a during proteolytic stress but not during ER stress. Furthermore, deletion of Herp enhanced the amount of ubiquitinated protein in the cytosol during proteasomal inhibition, although it did not affect the activity or expression of proteasome. These results suggest that ERAD molecule Herp may delay the degradation of cytosolic proteins at the ubiquitination step.     

Mutational and expressional analyses of SPOP,a candidate tumor suppressor gene,in prostate,gastric and colorectal cancers

Mounting evidence exists that alterations of ubiquitination processes are involved in cancer pathogenesis. Speckle‐type POZ protein (SPOP) is a key adaptor for Cul3‐based ubiquitination process. Recent studies reported that SPOP may be a tumor suppressor gene (TSG) and somatic mutation of SPOP was detected in prostate cancer (PCA). The aim of this study was to see whether alterations of SPOP protein expression and somatic mutation of SPOP gene are features of cancers. In this study, we analyzed SPOP somatic mutation in 45 gastric (GC), 45 colorectal cancer (CRC) and 45 PCA by single‐strand conformation polymorphism (SSCP). Also, we analyzed SPOP protein expression in 60 GC, 60 CRC and 60 PCA by immunohistochemistry. Overall, we detected three somatic missense mutations of SPOP gene in the coding sequences (p.Ser14Leu, p.Tyr87Cys and p.Phe133Leu). The mutations were observed in two PCA and one CRC. Of note, the p.Phe133Leu was a recurrent mutation reported in an earlier study. In the immunohistochemistry, SPOP protein was expressed in normal gastric, colonic and prostate epithelial cells, whereas it was lost in 30% of GC, 20% of CRC and 37% of PCA. Our data indicate that loss of SPOP expression was common in GC, CRC and PCA, but somatic mutation of SPOP in this study was rare in these tumors. Also, the data provide a possibility that loss of expression of SPOP gene might play a role in cancer pathogenesis by altering TSG functions of SPOP.     

The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor

We previously identified the E3 ubiquitin ligase IDOL as a sterol-dependent regulator of the LDL receptor (LDLR). The molecular pathway underlying IDOL action, however, remains to be determined. Here we report the identification and biochemical and structural characterization of an E2–E3 ubiquitin ligase complex for LDLR degradation. We identified the UBE2D family (UBE2D1–4) as E2 partners for IDOL that support both autoubiquitination and IDOL-dependent ubiquitination of the LDLR in a cell-free system. NMR chemical shift mapping and a 2.1 Å crystal structure of the IDOL RING domain–UBE2D1 complex revealed key interactions between the dimeric IDOL protein and the E2 enzyme. Analysis of the IDOL–UBE2D1 interface also defined the stereochemical basis for the selectivity of IDOL for UBE2Ds over other E2 ligases. Structure-based mutations that inhibit IDOL dimerization or IDOL–UBE2D interaction block IDOL-dependent LDLR ubiquitination and degradation. Furthermore, expression of a dominant-negative UBE2D enzyme inhibits the ability of IDOL to degrade the LDLR in cells. These results identify the IDOL–UBE2D complex as an important determinant of LDLR activity, and provide insight into molecular mechanisms underlying the regulation of cholesterol uptake.     

RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex

Rhobtb2 is a candidate tumor suppressor located on human chromosome 8p21, a region commonly deleted in cancer. Rhobtb2 is homozygously deleted in 3.5% of primary breast cancers, and gene expression is ablated in approximately 50% of breast and lung cancer cell lines. RhoBTB2 is an 83-kD, atypical Rho GTPase of unknown function, comprising an N-terminal Rho GTPase domain and two tandem BTB domains. In this report, we demonstrate that RhoBTB2 binds to the ubiquitin ligase scaffold, Cul3, via its first BTB domain and show in vitro and in vivo that RhoBTB2 is a substrate for a Cul3-based ubiquitin ligase complex. Moreover, we show that a RhoBTB2 missense mutant identified in a lung cancer cell line is neither able to bind Cul3 nor is it regulated by the ubiquitin/proteasome system, resulting in increased RhoBTB2 protein levels in vivo. We suggest a model in which RhoBTB2 functions as a tumor suppressor by recruiting proteins to a Cul3 ubiquitin ligase complex for degradation.     

Jeremy Jass Prize for Research Excellence in Pathology 2013

Emerging evidence indicates that Cullin 4B (CUL4B), a major component of ubiquitin ligase complexes, is over‐expressed in diverse cancer types with pro‐tumourigenic effects. In this issue of the Journal of Pathology, Yuan and colleagues [6] elucidated the oncogenic activity of CUL4B in hepatocellular carcinoma (HCC) and delineated its role in driving Wnt/β‐catenin signalling. In addition to the stabilization of β‐catenin protein against proteasomal degradation, CUL4B also acts in concert with enhancer of Zeste homologue 2 (EZH2) to concordantly silence multiple Wnt inhibitors. These findings provide significant mechanistic insights into the epigenetic activation of the Wnt/β‐catenin pathway in HCC and shed light on the functional importance of ubiquitination in this intricate regulatory system. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Midori‐ishi Cyan/monomeric Kusabira‐Orange‐based fluorescence resonance energy transfer assay for characterization of various E3 ligases

Many bacterial pathogens hijack the host ubiquitin system for their own benefit by delivering effectors with ubiquitin ligase (E3) into host cells via the type III secretion system. Therefore, screening for small compounds that selectively inhibit bacterial but not mammalian E3 ligases is a promising strategy for identifying molecules that could substitute for antibiotics. To facilitate high‐throughput screening for bacterial E3 ligase inhibitors, we developed a MiCy/mKO (Midori‐ishi Cyan/monomeric Kusabira‐Orange)‐based FRET (fluorescence resonance energy transfer) assay and validated it on Shigella IpaH E3 ligase effectors. We showed the feasibility of using the MiCy/mKO‐based FRET assay to identify the most appropriate ubiquitin‐conjugating enzymes (E2s) and determine the lysine specificity of a given E3, both hallmarks of E3 activity. Furthermore, we showed the usefulness of the FRET assay in characterizing mammalian E3 ligases, such as TNF receptor‐associated factor 6 (TRAF6) and mouse double minute 2 homologue (MDM2). In addition, we confirmed the feasibility of determining the efficiency of inhibition of E3 ligase activity using inhibitors of E1 ubiquitin‐activating enzymes, such as UBE1‐41, by measuring the IC₅₀. Based on these results, we concluded that the MiCy/mKO‐based FRET assay is useful for characterizing E3 enzyme activity, as well as for high‐throughput E3 inhibitor screening.     

PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex

The DNA polymerase delta processivity factor Proliferating Cell Nuclear Antigen (PCNA) promotes the DNA damage-induced degradation of the replication initiation factor Cdt1 via the CRL4(Cdt2) E3 ubiquitin ligase complex. Here we demonstrate that PCNA promotes the ubiquitylation and degradation of the CDK inhibitor p21 in cells irradiated with low dose of ultraviolet (UV) by a similar mechanism. Human cells that are depleted of Cul4, DDB1 (damage-specific DNA-binding protein-1), or the DCAF Cdt2, are deficient in the UV-induced ubiquitylation and degradation of p21. Depletion of mammalian cells of PCNA by siRNA, or mutations in p21 that abrogate PCNA binding, prevent UV-induced p21 ubiquitylation and degradation, indicating that physical binding with PCNA is necessary for the efficient ubiquitylation of p21 via the CRL4(Cdt2) ubiquitin ligase. Cdt2 functions as the substrate recruiting factor for p21 to the rest of the CRL4 ubiquitin ligase complex. The CRL4(Cdt2) E3 ubiquitin ligase ubiquitylates p21 both in vivo and in vitro, and its activity is dependent on the interaction of p21 with PCNA. Finally, we show that the CRL4(Cdt2) and the SCF(Skp2) ubiquitin ligases are redundant with each other in promoting the degradation of p21 during an unperturbed S phase of the cell cycle.     

SCF(beta-TrCP1) controls Smad4 protein stability in pancreatic cancer cells

Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 protein degradation in pancreatic cancer cells. We found that F-box protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.     

Identification of a novel telomerase repressor that interacts with the human papillomavirus type-16 E6/E6-AP complex

The critical immortalizing activity of the human papillomavirus (HPV) type-16 E6 oncoprotein is to induce expression of hTERT, the catalytic and rate-limiting subunit of telomerase. Additionally, E6 binds to a cellular protein called E6-associated protein (E6-AP) to form an E3 ubiquitin ligase that targets p53 for proteasome-dependent degradation. Although telomerase induction and p53 degradation are separable and distinct functions of E6, binding of E6 to E6-AP strongly correlated with the induction of hTERT. Here, we demonstrate using shRNAs to reduce E6-AP expression that E6-AP is required for E6-mediated telomerase induction. A yeast two-hybrid screen to find new targets of the E6/E6-AP E3 ubiquitin ligase complex identified NFX1. Two isoforms of NFX1 were found: NFX1-123, which coactivated with c-Myc at the hTERT promoter, and NFX1-91, which repressed the hTERT promoter. NFX1-91 was highly ubiquitinated and destabilized in epithelial cells expressing E6. Furthermore, knockdown of NFX1-91 by shRNA resulted in derepression of the endogenous hTERT promoter and elevated levels of telomerase activity. We propose that the induction of telomerase by the HPV-16 E6/E6-AP complex involves targeting of NFX1-91, a newly identified repressor of telomerase, for ubiquitination and degradation.     

Structural and Functional Impact of Parkinson Disease‐Associated Mutations in the E3 Ubiquitin Ligase Parkin

Mutations in the PARKIN/PARK2 gene that result in loss‐of‐function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early‐onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure–function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.